Dataset Information

Recombinant cystatin-like protein-based competition ELISA for Trichinella spiralis antibody test in multihost sera.

ABSTRACT:

Objectives

Trichinella spiralis is a zoonotic parasite with a complex parasitic life cycle and exposed to animals or humans by infectious meat. To control transmissions of T. spiralis through the food chain to humans, sensitive and selective multihost sera-diagnosis is urgent needed for monitoring T. spiralis exposure.

Methods

A competition enzyme-linked immunosorbent assay (cELISA) for T. spiralis infection diagnosis in multihost sera was developed based on recombinant cystatin-like protein (rCLP-cELISA) as well as monoclonal antibodies. The sensitivity and accuracy of the rCLP-cELISA were quantified using swine (n = 1316), mice (n = 189) and human (n = 157) serum samples. T. spiralis-antibody targeting test ability of the rCLP-cELISA in swine (n = 22) and human (n = 36), instead of other parasites or viruses antibodies, was evaluated.

Results

The rCLP-cELISA showed high agreement with commercial ELISA kits in field swine sera assessed by Cohen's kappa value (κ = 0.7963). And it showed 100% specificity in human trichinellosis detection with sensitivity of 96.49%, no cross-reaction with other parasite or virus infections, and high positive detection rate of 87.5% in low-dose infected swine. Besides, the rCLP-cELISA exhibited potential in the detection of T. spiralis, T. nelsoni and Trichinella T8 infections.

Conclusions

The rCLP-cELISA can be used for T. spiralis-associated antibody test in multihost sera.

SUBMITTER: Liu Y

PROVIDER: S-EPMC8423253 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Publications

Recombinant cystatin-like protein-based competition ELISA for Trichinella spiralis antibody test in multihost sera.

Liu Yan Y Xu Ning N Li Yansong Y Tang Bin B Yang Hualin H Gao Weihua W Liu Mingyuan M Liu Xiaolei X Zhou Yu Y

PLoS neglected tropical diseases 20210825 8

<h4>Objectives</h4>Trichinella spiralis is a zoonotic parasite with a complex parasitic life cycle and exposed to animals or humans by infectious meat. To control transmissions of T. spiralis through the food chain to humans, sensitive and selective multihost sera-diagnosis is urgent needed for monitoring T. spiralis exposure.<h4>Methods</h4>A competition enzyme-linked immunosorbent assay (cELISA) for T. spiralis infection diagnosis in multihost sera was developed based on recombinant cystatin-l ...[more]

PMID: 34432796

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Project description:Trichinella spiralis can modulate host immune responses to retain a suitable environment for its long-term survival. Incidentally, the parasite elicits regulatory effects through immunomodulatory molecule release, which can suppress host inflammation and may be used for the treatment of unrelated inflammatory diseases in someday. Here we identified and characterized a novel T. spiralis cystatin (TsCstN), which inhibits inflammation mediated by LPS-treated macrophages.Proteins contained in the excretory-secretory (ES) product of muscle-stage T. spiralis (ES-L1) were fractionated, and each was treated with mouse bone marrow-derived macrophages (mBMDMs) before LPS stimulation. The fractions that exhibited high immunomodulatory property by decreasing pro-inflammatory cytokines or increasing anti-inflammatory cytokines were identified by mass spectrometry. Incidentally, the conserved hypothetical protein (Tsp_04814) was selected for further characterization as it presented the most significant MS score. An annotation of Tsp_04814 using protein structural homology comparison suggested that it has high structural similarity to human cystatin E/M (TM score 0.690). The recombinant T. spiralis novel cystatin (rTsCstN) was expressed in Escherichia coli at a molecular weight of approximately 13 kDa. Mouse anti-rTsCstN polyclonal antibody (pAb) could detect native TsCstN in crude worm antigens (CWA) and ES-L1 and be predominantly localized in the stichosome and subcuticular cells. rTsCstN inhibited cysteine proteases in vitro, especially cathepsin L, at an optimal pH of 6. Besides, rTsCstN could be internalized into mBMDMs, which were mostly distributed in the cytoplasm and lysosome both before and after LPS stimulation. To evaluate the rTsCstN immunomodulatory properties on mBMDMs, rTsCstN was incubated with mBMDM before LPS stimulation; this demonstrated that rTsCstN suppressed pro-inflammatory cytokine production and MHC class II expression.T. spiralis L1-derived TsCstN was characterized as a novel cysteine protease inhibitor. The protein elicits an anti-inflammatory property by suppressing pro-inflammatory cytokines and interfering with the antigen presentation process through depletion of MHC class II expression.

Project description:The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T. spiralis and T. britovi recognized by Trichinella-infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti-Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T. spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T. britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T. spiralis and 18 for T. britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01_7775 and P49 antigen, partial those specific to T. spiralis; deoxyribonuclease-2-alpha and hypothetical protein T03_17187/T12_7360 were specific to T. britovi. Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species.

Project description:Trichinellosis is a zoonotic disease caused by the ingestion of the Trichinella nematode. With a worldwide incidence of approximately 10,000 cases per year, Trichinella spiralis is responsible for most human infections. There are no specific signs or symptoms of this parasitic infection. Muscle biopsy is the gold diagnostic standard for trichinellosis, but the technique is invasive and unable to detect the early stage of infection. Although immunodiagnostics are also available, antibody detection usually occurs after 3 weeks and prolonged up to 19 years after the acute phase. Therefore, additional diagnostic biomarkers must be identified to improve trichinellosis diagnosis. This study aimed to measure concentration changes in mouse serum proteins prior to T. spiralis infection and 2, 4 and 8 weeks after infection, and to identify T. spiralis circulating proteins and antigens using mass spectrometry-based proteomics. Mouse muscle-related proteins including inter-alpha-trypsin inhibitor heavy chain H2, a protein involved in the response to muscle tissue damage, were up-regulated in mouse sera during the T. spiralis larvae invasion. Additionally, 33 circulatory parasite proteins were identified in infected mouse sera. Notably, T. spiralis long-chain fatty acid transport protein 1 could be detected in the early stage of infection and peroxidasin-like protein was identified 2, 4 and 8 weeks after infection. Seventeen T. spiralis circulating antigens were detected in mouse immune complexes, with PX domain protein being found 2, 4 and 8 weeks after infection. Because peroxidasin-like protein and PX domain protein were detected at all post-infection time points, sequence alignments of these proteins were performed, which showed they are conserved among Trichinella spp. and have less similarity to the human and murine sequences. Integrative analysis of T. spiralis biomarkers throughout the course of infection may reveal additional diagnostic targets to improve early diagnosis of trichinellosis.

Dataset Information

Recombinant cystatin-like protein-based competition ELISA for Trichinella spiralis antibody test in multihost sera.

Objectives

Methods

Results

Conclusions

Publications

Recombinant cystatin-like protein-based competition ELISA for Trichinella spiralis antibody test in multihost sera.

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